Process of producing refined hog-cholera serum



- UNITED STATES PATENT OFFICE JOHN REICHEL, F PHILADELPHIA, PENNSYLVANIA, ASSIGNOR TO K. MULFORD COMPANY, OF PHILADELPHIA, PENNSYLVANIA.

rRocEss or rnonncine REFINE!) HOG-CHOLERA SERUM.

No Drawing.

To all whom it may concern: I

Be it known that I, JOHN REIOHE a citizen of the United States, residing in Philadelphia, Pennsylvania, have disi covered a certain new and useful. Process from hog cholera antitoxinin the form of serum or defibrinated blood by the use of chemical precipitants other than those pro ducing insoluble hydroxide and has for an object to obtain an improved hog cholera antitoxin containing the hog cholera immune, or anti-bodies, and the hog cholera globulin with which hogs may be immunized against hog cholera, and hogs sick with hog cholera may be cured. V H

In carrying out the process of'the discovery hog cholera antitoxin may be used in the form of serum or ,defibrinatedblood, either fresh, sterile, preserved or otherwise, and therefor the process in practice is not to be considered as restricted to any particular prepared form of hog cholera antitoxin. Injections of virulent blood or virus, from a hog sick of hog cholera or the cause of hog cholera, into an animal immune'from hog cholera from any cause, produces in the.

- blood of that immune animal an antitoxin or hog cholera immune bodies which may be obtained or drawn ofiiln the form of a serum or defibrinated blood containing many substances aside from the purely .Watery portion, such as globulins, serum Y albumins, cellular debris, fibrin, living or dead germs and salts, among which the hog cholera immune bodies are included as a part or are closely associated therewlth.

It is to the antitoxin obtained in this or;

other ways thatv this. process relates more particularly, and the process is carried out by the following treatment steps in order;

to remove the living or dead germs,

. cellular debris, fibrin, serumalbumin, and

recover the hog cholera immune or antitoxin bodies along with the globulins or hog cholera .serum globulin. The process consists primarily of breaking up the serum into a number of fractions, ineach of which there iscontained some ofthe desired hog ,diluent or dialyzed, ready for use."

Specification of Letters Patent. Patented Apr, 6 1920, Application filed April 3, 1914. Serial No. 829,176. r i V cholera immune bodies and globulins, and these may be separated or recovered from either fraction to obtain the hog cholera serum globulin in solution or powder, rich. or poor 1n hog cholera immune bodies acthe several fractions contain "different percording to the fraction treated provided centages of the immune bodies and globu line, which latter condition has been found by experimentation as the more general one The process is preferably carried out by taking a definite amount of'hog cholera antitoxin, serum or defibrinated blood, as above described, and treating it with a chemical precipitant, other than those form- The 1 (pScmdmgbbuhH'S and euglobulins'), which is then filtered and removed from the solution and soluble contents;

' The filtrate resulting from this step is a solution comparatively rich in hog cholera immune bodies and psuedo globulins, and

may be termed for convenience the B. fraction, which is preferably treated separate from the A. fraction to obtain its active constituents.

This is accomplished. by bringing.

the filtrate or B fraction up to higher saturation of the chemical, in this instance ammonla sulfate, giving substantially a 50% solution of the chemical. The precipiand psuedo golbulins. This B. fraction precipitate is filtered and may then be quickly washed, dried, powdered, dissolved i i a he chemicalreagents used in the process may be. removed partially or entirely from the B fraction precipitate by absorption, or dialyses, and the hog cholera immune bodies Y tate from this solution contains'a large percentage of the hog cholera mmune bodies and globulins,'partially or entirely. in 0111, tion, may then be preserved by filtration or j addition of a preservative. V p p Returning, now, to the A fraction .precie 'pitate, the hog cholera immune bodies and ,globulins contained therein may be sepa;

sodium chlorid filtrate are then precipitated by the addition of a suitable chemical precipitant, such as glacial acetic acid and the precipitate removed by filtration.

The resultant globulin precipitates from the respective fractions A and B may be added one to the other, or each may be used alone in the preparation of hog cholera antitoxin from which the living and dead germs, cellular debris, fibrin and serum alhumin have been eliminated and in connection with in ect1ons of hog cholera virus ,or the cause of hog cholera to prevent the disease known as hog cholera in healthy hogs, and to cure hogs sick of hog cholera.

While the process above described is made up of a number of steps and in eliminating living or dead germs, cellular debris, fibrin, serum albumin from hog cholera antitoxin and for specifically recovering from hog cholera antitoxin, the hog cholera immune bodies and globulins or active substances of the A and B fractions, it is to be understood that the process is not to be limited to the specific number of steps described, sincebroadly considered the process is adequately embodied in those steps constituting the recovery of the active substances, hog cholera immune bodies and 'globulins or hog cholera serum globulin from the B fraction, and obviously the process in its specific details 7 may consist of substantially an indefinite number of fractional recoveries of the active" substances, and therefore the scope of the process is not restricted in any manner tothe specific details here recited.

Attention is particularly directed to the fact that the chemical precipitants used are not such as to form insoluble hydroxids, and while mention is made of ammonia sulfate, sodium chlorid and glacial acetic acid, it is not to be understood that the new process is limited to theuse of these chemicals since they have been mentioned only by way of example and as one means of carrying,

out the process. Furthermore it will be ,noted that the fractional precipitationof the hog cholera immune bodies and globulins or active substances from the hog chol- V era antitoxin, either fresh, sterile, preserved or otherwise, by "which the latter is freed from living or dead germs, cellular debris, fibrin, or serum albumin, is accomplished without the 'useof heat.

The nature of the resultant hog cholera I claim and serum obtained by the process of this dis covery may be more readily understood and its advantages appreciated when it, is stated that the ordinary commercialhog cholera antitoxin supplied and used in the present day practice is a comparatively heavy turbid liquid of reddish hue containing in addition to the active substances, serum albumin, cellular debris, fibrin, living or dead germs, some of which are even perceptible to the eye in the product as ordinarily pre pared. When this commercial hog cholera antitoxin is introduced or injected intoa hog suffering from hog cholera or into a healthy cholera-free hog to prevent the development of hog cholera, it frequently occurs that the germs introduced along with the active substances offsetwhatever beneficial effect is anticipated by the active substances.

still further disadvantage inherent in the ordinary hog cholera antitoxin resides in the difiiculty with which absorption occurs when injected into an animal under treatment. It is a frequent occurrence when injecting a hog with hog cholera 'antitoxin, to

subsequently find the unabsorbed portion of the antitoXin at or near the point ofinjection in the form of a cold encapsulated or 7 active abscess, all of which more or less limits the possible value of the hog cholera antitoxin. I T

By the present process hog cholera serum globulin, an entirely new substance as far as I am aware, may be obtained, and is avail able as a clear, substantially transparent 'solution, entirely free of living or dead germs,

cellular debris, fibrin or other inert substances and also lacking in the presence of r the serum albumin. 1

It will now'be apparent that'the process embodies thefeatures of advantage enumerated, as desirable in the statement of the discovery and in the above description, and while in the present instance the preferred embodiment hasbeen described, which gives I satisfactory and reliable results, it is to be understood that the same is susceptible of modification in various particulars without departing from thespirit or scope of the discovery or sacrificing any of its advantages.

Having thus describedmy invention, what desire to protect by Letters Patent is: V t J 1. Tlie process of eliminating from hog cholera .ahtitoxin, the serum albumins, cellular deb'ri'sffibrin, or living or dead germs, by precipitation with'chemical .pre-

c1 itant oth r than those forming insoluble hydroxids'and filtration andpr'eservi'ng only the active substances, whereby the hog cholera immune bodies andthe globulins are ohtained having the characteristics of increasing the resistanee of'the hog against infec tion from hog cholera virus 'orthecause of hog cholera, and adapted to be used either only the activesubstances, whereby the hog cholera immune bodies and the globulins are obtained having the characteristics of increasing the resistance of the hog against in fection from hog cholera virus or the cause of hog cholera, and adapted to be used either alone or in connection with hog cholera virus or the cause of hog cholera to prevent the'disease known as hog cholera in healthy hogs or to cure hogssick of hog cholera.

3. In the process of producing hog cholera globulin from hog cholera antitoxin, defibrinated blood or serum, the step which consists, in treating it with ammonia sulfate,

so that the resulting mixture contains approximately thirty-three and a' third per cent. of the chemical and filtering the mixture.

4-. in the process of producing hog cholera globulin from hog cholera f antitoxin', defibrinated blood or serum, the steps which consist, in treating it with ammonia sulfate, so that the resulting mixture containsapproximately thirty-three and one third per cent. of the chemical, obtaining a filtrate from the mixture, treating the filtrate with ammonia sulfate, so that the resulting mixture contains substantially a fifty per cent.-

solution of the chemical, and filtering the precipitate.

5. The process of producinghog cholera glol'nilin from hog cholera antitoxin, defibrinate-d blood or serum, the steps which consist, in treating it with ammonia sulfate, so that the resulting mixture contains approximately thirty-three and a third per cent. of the chemical, obtaining a filtrate frmn the mixture, treating the filtrate with ammonia sulfate, so that the resulting mixture contains substantially a fifty per cent. solution of the chemical, and filtering the precipitate and partially or entirely remov ing from the precipitate the chemicals used in the said steps.

6. The process of producing hog cholera immune bodies and globulin from hog cholera antitoxin, defibrinated blood or serum, the steps which consist, in treating it with ammonia sulfate, so that the resulting mix ture contains approximately thirty-three and a third per cent. of the chemical, obtain ing a filtrate from the mixture, treating the filtrate with ammonia sulfate, so that the resulting mixture contains substantially a fifty per cent. solution of the chemical, and filtering the precipitate, and partiallyor entirely removing from the precipitate the chemicals used, and'then preservingthe hogcholera' immune bodies and globulins by filtration or the addition of a preservative.

V 7. The process of producing hog cholera globulin from hog cholera. antitoxin, de-

fibrinated blood .or serum, the step which consists, in treating it with ammoniasulfate, so that the resulting mixturecontains approximately thirty-three and a third per cent. of the chemical, obtaining a precipitate froin the mixture, macerating the precipitate in water, removing therefrom by'filtration the insoluble cellular debris, fibrin and killed germs, addin sodium. chlorid to the filtrate,

until thefiltrate is saturated with the chemical, removing; the precipitate therefrom, adding to the preclpitate glac al acetic acld, and removing the precipitate by filtration.-

8. The process of producing hog cholera globulin, which consists in mixing hog cholera ant1tox1n',. defibrinated blood or serum with ammonia sulfate, so that the resulting mixture contains approximately. thirty-three and one third. per cent. of the chemical, 0btaining froin the mixture a,filtr ate, treating the filtrate with ammonia sulfate,- so that the resulting mixture contains substantially a fifty per cent. solution of the chemical, and filtering, and washing, drying, powdering,

dissolving in a diluent or dialyzing the preand curing those artificiallyinfected with hog cholera, and-adapted to-be used either alone or in connection with injections of-the virus of hog cholera, to prevent the-disease known as fhog choleradin healthy hogs,

and to curehogs sick of hog cholera.

10. A substance produced fromhog cholllOT era defibrinatedblood antitoxin, by treat ing the same with a chemical precipitant and filtering, the filtrate possessing the active substance thereof, to prevent the disease known, as hog cholera in healthy hogs,- and to cure hogs sick'of hog cholera, and having the characteristics of being practically'fre'ed of living or dead germs, cellular debris or fibrin. V I

1.1. A substance produced from hog cholera defibrinated blood antitoxin and possess ing the active substances thereof, to prevent the disease known as hog cholera in healthy hogs, and to cure hogs sick of hog cholera, and having the characteristics of being in a concentrated form, and practi .cally freed of living or dead germs, cellular debris, fibrin or serum albumin.

12. A new substance, concentrated hog or defibrinated blood, and having the characteristics of immunizing hogs against in fection from hogcholera and curing those artlficially infected wlth hog cholera, and

" adapted to be used either alone or in connection with injections of the virus of hog cholera, to prevent the disease known as ho cholera 1n health he s and to cure D 7 hogs s1ck of hog cholera.

13.. A substance produced from hog-cholera defibrinated blood antitoxin and possessing the active substances thereof, and

7 having the characteristics of being in a sterile form, and freed of living or dead germs, cellular debrls', fibrln or serum albumin, the same being held in solution or suspens1on or being 1n solid form as a powder capable of solution or suspension.

'ltaA substance produced from and possessing the active substances of hog-cholera defibrinated blood antitoxin to prevent the disease known as hog cholera in healthy hogs, and to cure hogs sick of hog cholera, and having the characteristics of being in .7 a concentrated and sterile form, and freed of living or dead germs, cellular debris,

fibrin or serum albumin. 1

. 15. A new substance produced from hogcholera defibrinatedblood antitoxin, consisting of the hog-cholera immune bodies and the globulins, having the characteristics of being in sterile and concentrated form, and increasing the resistance of the hog against infection from hog cholera virus or the cause of'hog cholera and adapted tobe used either alone or in'connection with'hog cholera virus or the cause of hog cholera to preventthe disease known as hog cholera in healthy hogs and to cure hogs sick of hog cholera.

' sisting of the hog-cholera immune bodies 16. A new substance produced from hogcholera 'defibrinated blood antitoxin, conand the globulins, having the characteristics ofbeing in sterile and concentrated form,

and held in solution of suspension or being in solid form as a powder capablerof solution or' suspensioinand 'of increasing the resistance of the hog against infection from V hog cholera virus or the cause of hog cholera and adapted to be used either alone or in connection with hog cholera virus or the cause of hog cholera to prevent the disease known as hog cholera in healthyh'ogs'andv to cure hogs sickof hog cholera. I

17. A new substance made from hog cholera defibrmated blood antitoxinand consisting of the hog cholera globulins, and

possessing the characteristics of being in sterile form, and increasing the resistance of the hog against infectionfrom hog cholera virus or the cause of hog chol'era and adapted to be used either alone or in connection with hog cholera virus or the cause of hog cholera to prevent the disease known as hog cholera in healthy hogs, and to cure hogs sick of hog cholera.

' 18. A new substance made from hog cholera defibrinated blood antitoxin, consisting of the hog cholera pseudo-globulins, and practically freed from living or dead germs, cellular debris, fibrin, or serum albumin,

possessing the characteristics of, increasing:

the resistance of the hog against infection from hog cholera virus or the'cause of hog cholera and adapted to be used either alone or in connection with hog cholera virus or the cause of hog-cholera to prevent the disease known as hog cholera in healthy hogs, and to cure'hogs sick of hogcholera. 19. As a new substance hog cholera globlulin, consisting only of the hog cholera immune, bodies and the globulins obtained from hog cholera defibrinated blood antitoxin, having the active substances in concentrated and sterile form. 20. A substance produced from hog chol 'era defibrinated blood antitoxin, and possessing the active substances thereof, and

having the characteristics of being in a sterile form, and freed of living or dead germs, cellular debris, fibrin or serum 91- bumin. V

In testimony that I claim the foregoing I as my discovery, I havehereunto signed my name this second day of April, 191%.

JOHN REICI-IEL.

In the presence of- HORACE D. Rnnvn, ROBERT M. BARR. 

